Nearly all forms of life, both plant and animal, either synthesize ascorbic acid (e.g., Vitamin C) or require it as a nutrient. Ascorbic acid was first identified to be useful as a dietary supplement for humans and animals for the prevention of scurvy. Ascorbic acid, however, also affects human physiological functions such as the adsorption of iron, cold tolerance, the maintenance of the adrenal cortex, wound healing, the synthesis of polysaccharides and collagen, the formation of cartilage, dentine, bone and teeth, the maintenance of capillaries, and is useful as an antioxidant.
For use as a dietary supplement, ascorbic acid can be isolated from natural sources, such as rosehips, synthesized chemically through the oxidation of L-sorbose, or produced by the oxidative fermentation of calcium D-gluconate by Acetobacter suboxidans. Considine, "Ascorbic Acid," Van Nostrand's Scientific Encyclopedia, Vol. 1, pp. 237-238, (1989). It is also known to obtain predominantly intracellular ascorbic acid through the fermentation of microorganisms of Chlorella pyrenoidosa. See U.S. Pat. No. 5,001,059 by Skatrud, which is assigned to the assignee of the present application. While not fully understood, it is believed that ascorbic acid is produced inside the chloroplasts of photosynthetic microorganisms and functions to neutralize energetic electrons produced during photosynthesis. Accordingly, ascorbic acid production is known in photosynthetic organisms as a protective mechanism.
The invention of the present application involves the novel use of microorganisms of the genus Prototheca to produce ascorbic acid. Organisms of the genus Prototheca are not photosynthetic and therefore would not have a need to produce ascorbic acid to function as a protective compound during photosynthesis. Early attempts at classifying organisms of the genus Prototheca identified the organism as a relation to photosynthetic Chlorella on the basis of morphology and life cycle. Modern references which mention such a relationship typically cite references from the early 1900s. For example, Lloyd et al., "The Cell Wall of Prototheca zopfii," J. Gen. Microbiol., 50:421-427 (1968) cites references from 1904, 1913 and 1927.
These early studies, however, were necessarily based on the analytical techniques available at that time. Today, however, Prototheca are not believed to be closely related to ascorbic acid producing species of Chlorella. Many modern references document numerous structural, biochemical and genetic differences between Chlorella and Prototheca.
With regard to structural differences between Prototheca and Chlorella, Webster et al. in "The Respiratory Chain of Colorless Algae, III. Electron Microscopy," J. Ultrastructure Research, 21:514-523, (1968), report (1) the presence of multiple Golgi bodies in Prototheca, whereas Chlorella has a maximum of one Golgi body, (2) the presence of vacuoles in Prototheca which are absent in Chlorella, (3) the positioning of mitochondria near the cell walls in Prototheca compared to the positioning of mitochondria in the cup of the chloroplast in Chlorella, (4) the cell wall of Prototheca consists of several layers, whereas the cell wall appears to be one layer in KMnO.sub.4 -fixed material for Chlorella, (5) the cell wall pulls away from the cytoplasm in KMnO.sub.4 -fixed material for Chlorella which does not occur for Prototheca, (6) the inward movement of the inner material of the cell wall during division in Prototheca which has not been observed in Chlorella, and (7) the cell wall of Prototheca is more convoluted than that of Chlorella. Accordingly, Webster et al. conclude that the existence of these significant differences in ultrastructure makes the classification of Prototheca as a colorless Chlorella unlikely.
Similarly, Lloyd et al. in "The Cell Wall of Prototheca zopfii," J. Gen. Microbiol., 50:421-427 (1968), report differences in cell wall amino acid constituents of Prototheca and Chlorella, and that Prototheca have nodules on the surface of the cell walls which are absent in Chlorella. Accordingly, Lloyd et al. conclude their findings cast doubt on the relationship of Prototheca as a colorless Chlorella.
With regard to biochemical differences between the genera Prototheca and Chlorella, Casselton et al. in "Observations on the Nitrogen Metabolism of Prototheca Kruger," New Phytol., 68:731-749, (1968), have shown that Prototheca cannot utilize nitrate nitrogen as a nitrogen source, whereas Chlorella can, and further discuss that Prototheca are thiamine dependent, whereas Chlorella are not.
Likewise, Manners et al. in "The Molecular Structures of a Glucan and a Galactan Synthesized by Prototheca zopfii," Carbohydrate Research, 29:63-77, (1973), describe significant differences in polysaccharides synthesized by Prototheca and Chlorella. As a result, Manners et al. conclude that the overall results of their investigation would not support the view that Prototheca is simply the colorless counterpart of Chlorella.
With regard to genetic differences between Prototheca and Chlorella, Kerfin et al. in "Physiological and Biochemical Contributions to the Taxonomy of the Genus Prototheca, II. Starch Hydrolysis and Base Composition of DNA," Arch. Microbiol., 116:105-107, (1978), report that the DNA from a strain of Prototheca showed only 3.4% hybridization with labelled DNA from a strain of Chlorella vulgaris, which prompted Kerfin et al. to state that this finding indicates that Prototheca is not related to the typical Chlorellas. In addition, Kerfin et al. consider that the guanine and cytosine content of DNA between Chlorella and Prototheca is "quite different".
In summary, prior references which identify Prototheca as an achlorophyllous Chlorella are now viewed with considerable skepticism. Newer analytical techniques have shown such significant structural, biochemical and genetic differences between the two genera that such a relationship is highly unlikely.